Thymic stromal lymphopoietin (TSLP) promotes innate and adaptive pulmonary allergic inflammatory responses in mouse models of asthma, and human clinical trials reveal that TSLP has an important role in asthma pathophysiology. TSLP acts synergistically with IL-33 to activate lung group 2 innate lymphoid cells (ILC2) to produce IL-5 and IL-13, cytokines that are critical to the pathogenesis of allergic airway inflammation. There is also very strong evidence suggesting TSLP-activated ILC2 may be critical in the genesis and propagation of adaptive immunity-mediated allergic responses, and that TSLP may also support glucocorticoid resistant asthma. Despite the recognition that TSLP is a key cytokine in allergic airway inflammation and asthma, the precise molecular pathways that regulate lung TSLP expression and activation of IL-5/IL-13 producing ILC2 are not fully defined. A more complete description of the genes involved in TSLP expression and ILC2 activation may provide novel opportunities to better characterize the pathogenesis of allergic inflammation and identify drug targets capable of greatly reducing its severity. We hypothesize that there are multiple genes contributing to the regulation of the TSLP/ILC2 signaling axis that have not been identified in the context of this immunopathogenic pathway. To test this hypothesis, we have two specific aims. Specific aim 1 is to identify quantitative trait loci (QTL) that associate with TSLP expression by using the recombinant inbred mouse panel known as the Collaborative Cross (CC). The CC currently has 59 genetically distinct and fully inbred mouse strains that have been created from 8 laboratory and wild strains, with over 70,000 high-performing single nucleotide polymorphisms (SNP) available for use in precision mapping. Specific Aim 1 is supported by our novel preliminary data that reveal significant differences in airway and lung TSLP expression and the number of IL-5/IL-13 expressing ILC2 between three CC founder strains induced by airway challenge with an extract of Alternaria alternata, a fungal aeroallergen associated with severe asthma exacerbations. Upon obtaining phenotypic data available for all CC lines, we will use a haplotype reconstruction/regression statistical model to map QTL relevant to TSLP expression. For specific aim 2, we will determine the number of IL-5/IL-13 producing ILC2 as a phenotypic endpoint for each CC mouse. We will challenge mice with Alternaria extract for 4 consecutive days, and then harvest the following day to quantify the ILC2 response. We will repeat our statistical analysis to detect QTL relevant to ILC2 activation, hypothesizing that the genetic factors regulating these two related phenotypic endpoints are similar. QTL that we identify could be referenced to mouse genome banks that would allow us to identify biologically plausible genes for further investigation. The proposed experiment will advance the field in that it will provide new genetic candidates to study in the context of TSLP expression and ILC2 activation, allowing for a better mechanistic understanding of allergic inflammation pathogenesis.